Non-small cell lung cancer (NSCLC) is the major cause of cancer mortality in both women and men with a dismal <15% 5-year survival rate. Clearly, there is an urgent need for novel strategies to treat this disease. One strategy is to use the natural IGF binding protein, IGFBP-3, which regulates the insulin-like growth factor (IGF) pathway that plays a critical role in cell proliferation, antiapoptosis, survival, and neoplastic transformation. This proposal was based on preliminary data which have shown that: A) IGF pathway is selectively activated in NSCLC cells; B) targeting the IGF pathway selectively blocks the growth of NSCLC cells; C) IGFBP-3 has a dual role as a targeted therapeutic strategy for lung cancer treatment because it mediates IGF-independent intracellular antiproliferative and pro-apoptotic effects in addition to its IGF-dependent growth-regulatory function. The two separate pathways of IGFBP-3 may cooperate in an additive or synergistic manner to exert enhanced anti-tumor effects relative to each pathway alone; D) frequent loss of IGFBP-3 expression observed in samples from patients with stage I NSCLC were strongly associated with poor prognosis of stage I NSCLC One potential concern about the use of IGFBP-3 in lung cancer therapy is the presence of an activated protein kinase B (Akt) because we found that Akt induces the phosphorylation of IGFBP-3 leading to its degradation. Akt can be activated in NSCLC cells by IGF as well as by ras mutations and/or overexpression of members of EGFR family and their ligands, such as transforming growth factor alpha (TGF-alpha), which have been observed in 30-80% NSCLC patients. Because Akt can be activated via the Ras pathway even in cells treated with IGFBP-3 that blocks the IGF-mediated Akt activation, we hypothesize that agents, which inhibit IGF-independent Akt activity could enhance the antiproliferative effects of IGFBP-3 in NSCLC cells. Indeed, pharmacologic approach that inhibit Ras activation augmented IGFBP-3 protein level by increasing its stability and enhanced the growth inhibitory effects of IGFBP-3. This suggests that Akt plays a critical role in the function of cellular IGFBP-3 as an IGF-independent growth modulator in NSCLC cells. These and other studies have led to the following hypotheses: 1) IGFBP-3 inhibits lung cancer cell growth by virtue of its ability to bind to IGFs in addition to its IGF-independent effects. 2) Akt may inactivate IGF-independent growth-inhibitory function of IGFBP-3 by decreasing its stability due to phosphorylation in NSCLC cells. 3) Strategies to inhibit Akt activity in addition to suppressing IGF signaling will enhance the therapeutic effects of IGFBP-3 in lung cancer. In this revised RO1 application, I propose to test these hypotheses by exploring the following Specific Aims: 1) To determine whether the incidence and growth of lung tumors are inhibited by an increase in IGFBP-3 level and enhanced by loss of IGFBP-3 through the use of IGF-I transgenic mice and IGFBP-3 null mice. 2) To investigate a) whether inhibition of Akt activity will sensitize NSCLC cells to IGF-independent antiproliferative effects of IGFBP-3, and b) the mechanism through which the activation of Akt leads to NSCLC cell resistance to IGF- independent IGFBP-3 actions. 3) To explore the lung cancer therapeutic activity of a combined gene therapy approach using bicistronic adenoviral vector that inhibits the Akt activity and increases IGFBP-3 expression in IGF-I transgenic mice. We expect that the understanding the mechanism by which IGFBP-3 mediates signals for apoptosis and the mechanism that attenuates the antiproliferative effects of IGFBP-3 in NSCLC cells may lead to the development of more effective approaches to the treatment of lung cancer using IGFBP-3.